Diffuse Large B-Cell Lymphoma (“DLBCL”) is an aggressive and heterogeneous disease comprising at least three major subtypes with distinct molecular, biological, and clinical properties: activated B-cell-like (“ABC”) DLBCL, germinal center B-cell-like (“GCB”) DLBCL, and primary mediastinal B-cell lymphoma (“PMBL”) (Alizadeh et al., “Distinct Types of Diffuse Large B-cell Lymphoma Identified by Gene Expression Profiling,” Nature 403:503-511 (2000); Lenz et al., “Aggressive Lymphomas,” N. Engl. J. Med. 362:1417-1429 (2010); and Wright et al., “A Gene Expression-Based Method to Diagnose Clinically Distinct Subgroups of Diffuse Large B Cell Lymphoma,” Proc. Nat. Acad. Sci. U.S.A. 100:9991-9996 (2003)). Although the overall cure rate for DLBCL reaches over 50% with the current therapies, such as R-CHOP, fewer than 40% of ABC DLBCL patients are cured (Friedberg et al., “Diffuse Large B-Cell Lymphoma,” Hematol. Oncol. Clin. North Am. 22:941-952 (2008); Lenz et al., “Aggressive Lymphomas,” N. Engl. J. Med. 362:1417-1429 (2010); and Staudt et al., “The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling,” Adv. Immunol. 87:163-208 (2005)). Thus, new therapy approaches efficient for this subtype, as well as for other subtypes, are highly desirable.
The transcription factor NF-κB controls expression of a wide range of genes involved in cell proliferation, survival, stress response, angiogenesis, and inflammation (Hacker et al., “Regulation and Function of IKK and IKK-Related Kinases,” Sci. STKE rel3 (2006) and Hayden et al., “Shared Principles in NF-kappaB Signaling,” Cell 132:344-362 (2008)). NF-κB activity is tightly regulated by multiple signaling pathways, and abnormal NF-κB activation has been linked to cancer development and progression (Ben-Neriah et al., “Inflammation Meets Cancer, with NF-kappaB as the Matchmaker,” Nat. Immunol. 12:715-723 (2011); Karin, “Nuclear Factor-kappaB in Cancer Development and Progression,” Nature 441:431-436 (2006); Karin, “NF-kappaB as a Critical Link Between Inflammation and Cancer,” Cold Spring Harb. Perspect. Biol. 1:a000141 (2009); and Staudt, “Oncogenic Activation of NF-kappaB,” Cold Spring Harb. Perspect Biol. 2:a000109 (2010)). Constitutive NF-κB activation has been observed in high frequency in all main DLBCL subtypes, especially in ABC DLBCLs with more than 90% of the tumors showing nuclear NF-κB, which is the hallmark of its activation (Compagno et al., “Mutations of Multiple Genes Cause Deregulation of NF-kappaB in Diffuse Large B-cell Lymphoma,” Nature 459:717-721 (2009); Davis et al., “Chronic Active B-Cell-Receptor Signalling in Diffuse Large B-Cell Lymphoma,” Nature 463:88-92 (2010); Honma et al., “TNFAIP3/A20 Functions as a Novel Tumor Suppressor Gene in Several Subtypes of Non-Hodgkin Lymphomas,” Blood 114:2467-2475 (2009); Lenz et al., “Oncogenic CARD11 Mutations in Human Diffuse Large B Cell Lymphoma,” Science 319:1676-1679 (2008); Ngo et al., “Oncogenically Active MYD88 Mutations in Human Lymphoma,” Nature 470:115-119 (2011); and Staudt, “Oncogenic Activation of NF-kappaB,” Cold Spring Harb. Perspect Biol. 2:a000109 (2010)). A recent genomic study revealed that more than 60% of ABC DLBCLs and about 30% of GCB DLBCLs harbor somatic mutations in multiple components of NF-κB signaling pathways, such as B-cell receptor (“BCR”), CD40, and Toll-like receptor pathways (Pasqualucci et al., “Analysis of the Coding Genome of Diffuse Large B-Cell Lymphoma,” Nat. Gen. 43:830-837 (2011)). Importantly, it has been demonstrated that the constitutive NF-κB signaling is required for the proliferation and survival of ABC DLBCL cells lines (Davis et al., “Constitutive Nuclear Factor KappaB Activity is Required for Survival of Activated B Cell-Like Diffuse Large B Cell Lymphoma Cells,” J. Exp. Med. 194:1861-1874 (2001); Davis et al., “Chronic Active B-Cell-Receptor Signalling in Diffuse Large B-Cell Lymphoma,” Nature 463:88-92 (2010); Lam et al., “Small Molecule Inhibitors of Ikappab Kinase are Selectively Toxic for Subgroups of Diffuse Large B-Cell Lymphoma Defined by Gene Expression Profiling,” Clin. Cancer Res. 11:28-40 (2005); and Ngo et al., “Oncogenically Active MYD88 Mutations in Human Lymphoma,” Nature 470:115-119 (2011)). Collectively, these observations point to a primary role for constitutive NF-κB signaling in the pathogenesis of DLBCL. It is therefore proposed that the NF-κB signaling pathway may represent a rational therapeutic target in DLBCL (Rui et al., “Malignant Pirates of the Immune System,” Nat. Immunol. 12:933-940 (2011) and Staudt, “Oncogenic Activation of NF-kappaB,” Cold Spring Harb. Perspect. Biol. 2:a000109 (2010)).
Ubiquitination, the covalent attachment of ubiquitin (“Ub”) molecule to target proteins, regulates diverse cellular processes. Ubiquitination proceeds through a stepwise enzymatic cascade involving three classes of enzymes: a Ub-activating enzyme (“E1”), a Ub-conjugating enzyme (“E2”), and a Ub ligase (“E3”). The E1 enzyme activates ubiquitin in an ATP-dependent manner and transfers the activated ubiquitin to an E2 enzyme through the formation of a thioester bond between the carboxy terminus of ubiquitin and the active site cysteine of the E2, generating an E2 and Ub thioester conjugate (denoted as E2˜Ub). The E2 then cooperates with an E3 to attach the ubiquitin to a lysine residue of a substrate. Ubiquitin itself can serve as a substrate and the process can undergo multiple rounds, resulting in the formation of polyubiquitin chains (Pickart et al., “Ubiquitin: Structures, Functions, Mechanisms,” Biochim. Biophys. Acta. 1695:55-72 (2004); Weissman et al., “The Predator Becomes the Prey: Regulating the Ubiquitin System by Ubiquitylation and Degradation,” Nat. Rev. Mol. Cell Biol. 12:605-620 (2011); and Wenzel et al., “E2s: Structurally Economical and Functionally Replete,” Biochem. J. 433:31-42 (2011)). As ubiquitin has seven lysine residues and any one of them can be conjugated to another ubiquitin, polyubiquitin chains of different linkages with distinct functional properties are formed in cells. For example, lysine 48-(K48-) linked polyubiquitin chains typically target substrates for proteasomal degradation, while K63-linked polyubiquitin chains function as scaffolds to assemble protein complexes in DNA repair and NF-κB signaling (Liu et al., “Expanding Role of Ubiquitination in NF-kappaB Signaling,” Cell Res. 21:6-21 (2011); Panier et al., “Regulatory Ubiquitylation in Response to DNA Doublestrand Breaks,” DNA Repair 8:436-443 (2009); Wertz et al., “Signaling to NF-kappaB: Regulation by Ubiquitination,” Cold Spring Harb. Perspect. Biol. 2:a003350 (2010); and Ye et al., “Building Ubiquitin Chains: E2 Enzymes at Work,” Nat. Rev. Mol. Cell Biol. 10:755-764 (2009)).
Ubc13 (also known as UBE2N) is the active subunit of an E2 enzyme that catalyzes the synthesis of K63-linked polyubiquitin chains. It functions together with one of its two cofactors, UeV1A (UBE2V) and Mms2 (UBEV2), which are E2 variants that lack the active site cysteine residues (Wenzel et al., “E2s: Structurally Economical and Functionally Replete,” Biochem. J. 433:31-42 (2011) and Ye et al., “Building Ubiquitin Chains: E2 Enzymes at Work,” Nat. Rev. Mol. Cell Biol. 10:755-764 (2009)). In response to the engagement of several membrane receptors, such as T-cell receptor and toll-like receptors, Ubc13-UeV1A, in conjunction with the E3 enzyme TRAF6, catalyzes the formation of K63-linked polyubiquitin chains, which interact with both TAK1 and IKK complexes and thereby bring these two kinases into proximity. Consequently, the activated TAK1 phosphorylates and activates the IKK complex, which in turn phosphorylates IκB proteins, leading to IκB protein degradation and subsequent NF-κB activation (Liu et al., “Expanding Role of Ubiquitination in NF-kappaB Signaling,” Cell Res. 21:6-21 (2011) and Wertz et al., “Signaling to NF-kappaB: Regulation by Ubiquitination,” Cold Spring Harb. Perspect. Biol. 2:a003350 (2010)). In complex with the Mms2 cofactor, Ubc13 promotes the K63-linked ubiquitination at sites of DNA double-strand breaks, leading to the recruitment of repair proteins to the DNA lesions (Panier et al., “Regulatory Ubiquitylation in Response to DNA Doublestrand Breaks,” DNA Repair 8:436-443 (2009) and Ye et al., “Building Ubiquitin Chains: E2 Enzymes at Work,” Nat. Rev. Mol. Cell Biol. 10:755-764 (2009)). In addition to participating in NF-κB activation and DNA doublestrand break repair, Ubc13 regulates other cellular processes, including nuclear localization of tumor suppressor p53 protein and MAP kinase activation (Laine et al., “Regulation of p53 Localization and Activity by Ubc13,” Mol. Cell. Biol. 26:8901-8913 (2006); Topisirovic et al., “Control of p53 Multimerization by Ubc13 is JNK-Regulated,” Proc. Nat. Acad. Sci. U.S.A. 106:12676-12681 (2009); and Yamamoto et al., “Key Function for the Ubc13 E2 Ubiquitin-Conjugating Enzyme in Immune Receptor Signaling,” Nat. Immunol. 7:962-970 (2006)).
The chronic active BCR signaling, together with MYD88-mediated signaling, is largely responsible for the constitutive NF-κB activation in ABC DLBCL cells and controls the proliferation and survival of these cells. Hence, inhibition of this pathway is a target for new therapeutic strategies (Davis et al., “Chronic Active B-Cell-Receptor Signalling in Diffuse Large B-Cell Lymphoma,” Nature 463:88-92 (2010) and Rui et al., “Malignant Pirates of the Immune System,” Nat. Immunol. 12:933-940 (2011)). Indeed, small-molecule inhibitors of several kinases such as Syk and PKC-β, which mediate NF-κB activation in the BCR signaling pathway (Siebenlist et al., “Control of Lymphocyte Development by Nuclear Factor-kappaB,” Nat. Rev. Immunol. 5:435-445 (2005) and Thome et al., “Antigen Receptor Signaling to NF-kappaB via CARMA1, BCL10, and MALT1,” Cold Spring Harb. Perspect. Biol. 2:a003004 (2010)), have been tested in clinical trials with some efficacy (Dupire et al., “Targeted Treatment and New Agents in Diffuse Large B Cell Lymphoma,” Int. J. Hematol. 92:12-24 (2010)). As both Syk and PKCβ function upstream of the CARMA1 (CARD11)-BCL10-MALT1 (CBM) complex (Siebenlist et al., “Control of Lymphocyte Development by Nuclear Factor-kappaB,” Nat. Rev. Immunol. 5:435-445 (2005) and Thome et al., “Antigen Receptor Signaling to NF-kappaB via CARMA1, BCL10, and MALT1,” Cold Spring Harb. Perspect. Biol. 2:a003004 (2010)), inhibition of these kinases may have limited effect on the constitutive NF-κB activation in over 10% of DLBCLs that harbor mutations in the CBM complex (Compagno et al., “Mutations of Multiple Genes Cause Deregulation of NF-kappaB in Diffuse Large B-cell Lymphoma,” Nature 459:717-721 (2009); Lenz et al., “Oncogenic CARD11 Mutations in Human Diffuse Large B Cell Lymphoma,” Science 319:1676-1679 (2008); and Pasqualucci et al., “Analysis of the Coding Genome of Diffuse Large B-Cell Lymphoma,” Nat. Gen. 43:830-837 (2011)).
The present invention is directed to overcoming limitations in the art by providing new treatments for B cell malignancies.